Quantitation of cellular DNA and cell numbers using element labeling

ABSTRACT

Methods and kits for the quantitation of cellular DNA and cell numbers are provided. Passive element uptake, element-labeled DNA intercalators, and element labeled affinity reagents are used to quantify DNA and cells. The DNA and the cells are analyzed by elemental analysis, including ICP-MS. The methods and kits provide a fast and accurate analysis of cellular DNA and cell numbers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/772,589 filed Feb. 13, 2006, entitled “Quantitation of cellnumbers and cell size using metal labeling and elemental massspectrometry, the contents of which are herein incorporated by referencein their entirely.

COPYRIGHT AND LEGAL NOTICES

A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure, as it appears in the Patent and TrademarkOffice patent files or records, but otherwise reserves all copyrightswhatsoever.

FIELD

The invention relates to a rapid and sensitive assay employing elementalanalysis for cell enumeration and cell proliferation usingelement-labeled intercalators together with element-tagged affinityreagents to determine the cell type in a mixed cell population.

INTRODUCTION

“Intercalation” is functional non-covalent insertion of a ligand betweentwo molecular moieties. For example, the molecular moieties can benucleotide base units of double-stranded nucleic acid¹ or amino acids inproteins, to name a few. DNA intercalating reagents are extensively usedas sensitive molecular probes and therapeutic agents owing to their siteselective targeting and reactivity^(2;3). Metallointercalators areintercalating reagents having a transition-metal complex core.Metallointercalators can be bound by non-intercalating and intercalatingligands. For example, metallointercalators can be bound by twonon-intercalating ligands and one intercalating ligand. The intercalatorcan comprise other elements (i.e. non-metal elements, such as propidiumiodide with two iodide molecules) for DNA quantitation. “Elementalanalysis” is a process where a sample is analyzed for its elementalcomposition and sometimes isotopic composition. Elemental analysis canbe accomplished by a number of methods, including, but not limited to:I. optical atomic spectroscopy, such as flame atomic absorption,graphite furnace atomic absorption, and inductively coupled plasmaatomic emission, which probe the outer electronic structure of atoms;II. mass spectrometric atomic spectroscopy, such as inductively coupledplasma mass spectrometry, which probes the mass of atoms; III. X-rayfluorescence, particle induced x-ray emission, x-ray photoelectronspectroscopy, and Auger electron spectroscopy which probes the innerelectronic structure of atoms.

“Elemental analyzer” is an instrument functionally designed for thequantitation of atomic composition of a sample employing one of themethods of elemental analysis.

“Particle elemental analysis” is a process where an analyzed sample,composed of particles dispersed in a liquid (beads in buffer, forexample), is interrogated in such manner that the atomic composition isrecorded for individual particles (bead-by-bead, for example). Anexample of the analytical instrument is a mass spectrometer-based flowcytometer.

“Solution elemental analysis” is a process where an analyzed sample isinterrogated in such manner that the atomic composition is averaged overthe entire solution of the sample.

“Element tag” or “tag” is a chemical moiety which includes an elementalatom or multitude of elemental atoms with one or many isotopes attachedto a supporting molecular structure. In one instance an element tag maycomprise a metal-chelate polymer with an attachment group. Theattachment group may include, but is not limited to, pyrrole-2,5-dione(maleimido), sulfonic acid anion, or p-(chloromethyl)styrene (for thiol,N-terminus, or C-terminus, respectively). Other means are known to thoseskilled in the art.

The term “antibody” includes monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g. dual specificity antibodies),and antibody fragments, both natural and recombinant, as long as theyexhibit the desired biological activity or something functionallysimilar.

An “antigen specific antibody labeled with an element tag” comprises anantibody which has been subsequently reacted with an element tag whichallows the antigen-antibody complex to be detected and quantified bysolution elemental analysis.

An “affinity product” or “affinity reagent” refers to biologicalmolecules (antibody, aptamer, lectin, sequence-specific binding peptide,etc) which are known to form highly specific non-covalent bonds withrespective target molecules (peptides, antigens, small molecules, etc).Affinity reagent labeled with a unique element tag is an affinityproduct labeled with an element tag that is unique and distinguishablefrom a multitude of other element tags in the same sample.

A “transition element” means any element having the following atomicnumbers, 21-29, 39-47, 57-79, and 89. Transition elements include therare earth elements, lanthanides, and noble metals (Cotton andWilkinson, 1972).

An “internal standard” is defined as a known amount of a compound,different from analyte, that is added to the unknown. Signal fromanalyte is compared with signal from the internal standard to find outhow much analyte is present. An internal standard normally is used whenperforming mass spectrometry quantitation. An internal standard can alsobe used by other means known to those skilled in the art.

Fixing and permeabilization refers to chemical cross-linking of cellularcomponents by agents known to those skilled in the art, and may includebut not limited to glutaraldehyde, formaldehyde, formalin, ethanol,methanol, etc., and creating holes in the cell membrane with detergents.Suitable detergents may be readily selected from among non-ionicdetergents. These detergents may be used at concentrations between about0.001% to about 0.1%. One currently preferred detergent is TRITON™ X-100(Sigma T9284). Examples of other suitable detergents include Igepal andNonidet P-40. Other suitable detergents may be readily selected by oneof skill in the art.

It is accepted in cell biology that the content of nucleic acids is areasonable indicator of cell number owing to the tight regulation of DNAand RNA levels in the cells. DNA measurement is commonly used toestimate the number of cells in solid tumors as well as to characterizehematopoietic malignancies and monitor chemotherapy treatment^(4;5).Many studies demonstrate the prognostic significance of ploidy in humantumors. For example, trisomy 8 is one of the most frequent numericalchromosomal abnormalities observed in acute myelogenous leukemia (AML)and myelodysplastic syndrome (MDS)⁶. The content of DNA also reflectscell cycle progression. Cells grow through mitosis and the individualcells can be identified or classified, through their progression bymitosis, with a determination of the amount of DNA in the cell. Incombination with antigen identification, the measurement of DNAindicates the ploidy or cell cycle phase for a subset of cells definedby the antigen in heterogeneous cell populations, or the distribution ofa particular antigen through the cell cycle^(7;8).

Reagents that measure cell proliferation and cell numbers are importantdiagnostic and research tools. Standard methods of cell enumerationinclude BrdU (5-bromo-2′-deoxyuridine); ³H-thymidine incorporation intoreplicating cells during proliferation, and the measurement of totalnucleic acid content of lysed cells with a calorimetric (diphenylamine)reagent. Fluorescent DNA intercalating dyes in conjunction with flowcytometry have become the method of choice for rapid cell cycle and cellnumber measurements. Such DNA binding dyes as Hoechst 33258⁹, propidiumiodide¹⁰, DAPI¹¹ and acridine orange¹² have been shown in manyapplications to accurately estimate cell numbers. However, thesereagents suffer from relatively low fluorescence enhancements uponbinding nucleic acids, low extinction coefficients and have highintrinsic background fluorescence. Recently, a novel class of cyaninedyes and a sensitive nucleic acid stain-based assay have been developedby Molecular Probes Inc. which obviates these problems¹³.

Metallointercalators interact with double-stranded DNA throughelectrostatic forces—groove binding and intercalation, for example.Substantial research has been devoted to the photoluminescent¹⁴⁻¹⁶ andphoto-oxidizing properties of metallointercalators¹⁷⁻²¹.9,10-Phenanthrenequinone diimine (phi) complexes of rhodium(III)(Rh(III)) have been shown to bind tightly (Kd<10⁻⁸M) to double strandedDNA by intercalation in the major groove²²⁻²⁴ and undergo a variety ofphotoinduced reactions with DNA^(20;25-27). From photofootprintingexperiments Barton et al. (1992) concluded that Rh(phi)₂(bpy)³⁺ has arigid rhodium complex that can occupy directly all sites not obstructedby DNA binding proteins, and at rhodium/base pair ratios of 2:1 bindingto DNA is sequence neutral and random^(28;29). Recent work from the samegroup has also shown that metallointercalators which targeted majorgroove sites bind poorly to double-stranded RNA^(30;31). However, otherexperiments indicate that the apposition of several non-canonical basesas well as stem-loop junctions and bulges in tRNA could result inintimately stacked structures with opened major grooves accessible formetallointercalator binding³¹.

Recently, Inductively Coupled Plasma Mass Spectrometry (ICP-MS) has beenintroduced to the field of protein and cell surface antigenidentification via ICP-MS-linked immunoassays using metal containingimmunoreagents such as gold and lanthanide-conjugated antibodies³²⁻³⁵.ICP-MS as an analytical detector offers absolute quantification that islargely independent of the analyte molecular form or sample matrix.Secondly, the abundance sensitivity of ICP-MS, a measure of the overlapof signals of neighboring isotopes, is large (>10⁶ for the quadrupoleanalyzer), and this ensures independence of the detection channels overa wide dynamic range. The third key property is that ICP-MS is verysensitive; it was demonstrated that ICP-MS-linked immunoassays can be atleast as sensitive as radioimmunoassay.

ICP-MS is extensively used to study natural and induced metalincorporation into bacteria³⁶⁻³⁹, plants⁴⁰ and as a tool inmetalloproteomics research⁴¹.

The choice of the element to be employed in the methods of the presentinvention is preferably selected on the basis of its natural abundancein the sample under investigation and whether the element is cytotoxicto the sample under investigation.

Most metals of the transition and rare earth groups are anticipated foruse in passive uptake labeling of cultured cells. It is wise to chooseelements that have low or no cytotoxicity and have a low abundance ingrowth media and biological samples. For example, vanadium and mercurycan be toxic to certain cells, while Fe, Cu and Zn can be present inhigh concentrations in some cell culture media. On the other hand, Pr,Ho, Tb, La, for example are normally well tolerated by mammalian cellsand are not abundant in the environment.

Metallointercalators are synthesized using aromatic compounds as ligandsfor DNA binding and transition-metal complex cores. Metallointercalatorscan incorporate many types of metal centers such as Ru, Rh, Os, Co, Re,and Ir. A vast number of ligands are known to those skilled in the art(bipyridine, 9,10-phenanthrene quinone diimine, 1,10-phenanthroline,etc). Other element labeled DNA intercalators can also be used.

An unusual isotope composition of the tag element can be used in orderto distinguish between naturally present elements in the sample and thetag material. It is advantageous if the relative abundance of the tagelements is sufficiently different from the relative abundance ofelements in a given sample under analysis. By “sufficiently different”it is meant that under the methods of the present invention it ispossible to detect the target elemental tag over the background elementscontained in a sample under analysis. Indeed, it is the difference ininter-elemental ratios of the tagging elements and the sample matrixthat can be used advantageously to analyze the sample.

It is feasible to select elemental tags, which do not produceinterfering signals during analysis (i.e. do not have over-lappingsignals due to having the same mass). Therefore, two or more analyticaldeterminations can be performed simultaneously in one sample. Moreover,because the elemental tag can be made containing many copies of the sameatoms, the measured signal can be greatly amplified.

SUMMARY

These and other features of the applicant's teachings are set forthherein.

An aspect of the applicant's teachings is to provide a method toquantify the number of cells in a sample comprising cells, the methodcomprising: culturing the sample with an element to passively accumulatethe element in the cells in the sample; washing the cells to removenon-accumulated element; measuring the element in a known quantity ofthe cells by elemental analysis to determine the amount of accumulatedelement per cell; measuring the element in the sample by elementalanalysis; and quantifying the number of cells in the sample.

Another aspect of the applicant's teachings is to provide a method toquantify antigen content per cell in a cell sample wherein the cells ofthe sample comprise an antigen, comprising: culturing the cells of thesample in the presence of a first element wherein the cells passivelyaccumulate the first element; calculating the number of cells in thesample; removing non-accumulated first element; incubating the cellswith antigen specific affinity products labeled with a second elementtag; removing unbound affinity products; simultaneously measuring thepassively accumulated first element and the amount of the bound affinityproduct labeled with a second element tag by elemental analysis; andcalculating the antigen content per cell.

Another aspect of the applicant's teachings is to provide a method fordetermining the phase of a cell cycle in a sample of cells, comprising:determining the number of cells in the sample; fixing and permeabilizingthe cells; exposing the sample of cells to an element containingintercalator under conditions appropriate to incorporate theintercalator into the DNA within the cells of the sample. removingunbound intercalator; measuring the content of the labeled DNA withinthe cells by elemental analysis of the element; determining the amountof DNA per cell; and determining the phase of the cell cycle from theamount of DNA per cell.

Another aspect of the applicant's teachings is to provide a method todetermine DNA content and antigen content per cell in a sample of cells,comprising: determining the number of cells in the sample; fixing andpermeabilizing the cells; incubating the sample of cells with anintercalator tagged with a first element under conditions appropriate toincorporate the intercalator into the DNA within the cells of thesample; removing unbound intercalator from the cells; incubating antigenspecific affinity products labeled with a second element tag with thecells; removing unbound affinity products from the cells; simultaneouslymeasuring the intercalator and the affinity product labeled with thesecond element tag by elemental analysis; and calculating the DNAcontent and antigen content per cell.

Another aspect of the applicant's teachings is to provide a kit for thedetection and measurement of a first element in a sample, where themeasured first element originated from the first element passivelyaccumulated by cells, comprising: (a) a soluble compound of a firstelement for addition to a cell media. The kit can further compriseinstructions for (i) spiking a cell media with the soluble compound ofthe first element, ii) separating bound first element from unbound firstelement, and iii) detecting and measuring the passively accumulatedfirst element by elemental analysis. The kit can further comprise (a) asecond element for directly tagging an analyte specific affinityproduct; and (b) an analyte specific affinity product. The kit canfurther comprise instructions for (i) directly tagging the analytespecific affinity product with the second element; (ii) combining thetagged analyte specific affinity product with at least one type of ananalyte and analyte complex under conditions in which the tagged analytespecific affinity product binds with at least one of intra- or/andextra-cellular analyte and analyte complex, (iii) separating boundaffinity product from unbound affinity product, and iv) detecting andmeasuring the second element by elemental analysis. The kit can furthercomprise an analyte specific affinity product, wherein the analytespecific affinity product is directly tagged with a second element.

Another aspect of the applicant's teachings is to provide a kit for thedetection and measurement of a first element in a sample, where themeasured first element originated from a DNA intercalator, comprising:(a) a DNA intercalator comprising a first element. The kit can furthercomprising instructions for i) incorporating the first elementcontaining intercalator into the DNA within the cells of the sample, ii)separating bound intercalator from unbound intercalator, and iii)detecting and measuring the intercalator comprising the first element byelemental analysis. The kit can further comprise (a) a second elementfor directly tagging an analyte specific affinity product; and (b) ananalyte specific affinity product. The kit can further compriseinstructions for (i) directly tagging an analyte specific affinityproduct; (ii) combining the tagged analyte specific affinity productwith at least one type of an analyte and analyte complex underconditions in which the tagged analyte specific affinity product bindswith at least one of intra- or/and extra-cellular analyte and analytecomplex, (iii) separating bound affinity product from unbound affinityproduct, and iv) detecting and measuring the second element by elementalanalysis. The kit can further comprise an analyte specific affinityproduct wherein the analyte specific affinity product is directly taggedwith a second element.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. Upper plot—linear response between number of KG-1a cellspresent in each sample and amount of CD34 antigen detected as signalfrom Eu normalized to Ir internal standard (Eu/Ir) with backgroundsubtracted. Lower plot—relationship of KG-1a cell numbers andaccumulated metal, palladium, normalized to Ir internal standard(Pd/Ir).

FIG. 1B. Upper bar graph—mixed cell samples (1o, 2o, 3o, 4o, 5o, 7o onthe Y-axis) were incubated with negative control isotype specificimmunoglobulin and Eu-labeled secondary antibodies; analyzed for Eu, Pd,and Rh; signal values are relative to Ir internal standard. Lower bargraph—samples (#1, #2, #3, #4, #5, #7 on the Y-axis) were incubated withanti-CD34 primary and Eu-labeled secondary antibodies; analyzed for Eu,Pd, and Rh; signal values are relative to Ir internal standard.

FIG. 2. Linear concentration dependence between amounts of Rh(III)metallointercalator and signal obtained from treated cells (normalizedresponse).

FIG. 3. Normalized response for Rh obtained after treating live, orfixed, or fixed/permeabilized (fix/perm) MBA-1 cells with Rh(III)complex.

FIG. 4. Simultaneous identification of cellular DNA (Rh) and surfaceantigen CD33 (Eu) by solution elemental analysis. MBA-1 cells weretreated with Rh(III) complex and reacted with anti-CD33 antibodiesfollowed by Eu-labeled secondary antibodies. Sample #1 contains 3×10⁴,sample #2-3×10⁵, and sample #3-3×10⁶ cells.

FIG. 5. Detection of the internal Rh(III) labels (A) and the surface Aulabels (B) by particle elemental analysis with ICP-MS operated intransient mode. Single cell analysis of surface antigen CD34 expression(Au) and intracellular DNA (Rh) content were analyzed. KG-1a cells weretreated with intercalator and reacted with anti-CD34 antibodies followedby Au-labeled secondary antibodies.

FIG. 6. DNA content (Rh, normalized response) depending on cell cyclephase. K562 cells were grown under normal conditions (UN) or treated forsynchronization in the mitotic (M) or synthetic (G1/S) phase. Fixedcells were stained with Rh(III) complex and analyzed non-treated (non)or digested in Proteinase K (P) or RNAse A (R) solutions.

FIG. 7. Chart showing a method to quantify antigen content per cell, inaccordance with the invention.

FIG. 8. Chart showing a method for determination of cell cycle phase, inaccordance with the invention.

FIG. 9. Chart showing a method to determine DNA and antigen content percell, in accordance with the invention.

DESCRIPTION OF THE VARIOUS EMBODIMENTS

Aspects of the applicant's teachings may be further understood in lightof the following examples, which should not be construed as limiting thescope of the present teachings in any way.

Experiment 1

One embodiment can be demonstrated by example of rhodium (Rh) andpalladium (Pd) in mixed cell populations with surface marker identifiedby europium (Eu). The experiment was set up to investigate if metalspassively accumulated by cells could be indicative of cell number andcell type. A work flow chart is presented in FIG. 7. For this purposetwo different leukemia cell lines were cultured for 72 hours in growthmedia supplemented with very low amounts of rare earth metals: 100 ppbRh was added to MBA-1 cells, and 500 ppb Pd was added to KG-1a cellswhich express the cell surface marker. During the three days of cellculture, aliquots of cells were tested for viability with trypan blue.No excessive cell death was recorded due to the presence of metals inmedia. Cells were collected by centrifugation (1200 rpm, 10 minutes),washed once in PBS/10% FBS and counted in a hemocytometer. Serialdilutions were prepared to give 1×10⁶, 0.5×10⁶, 1×10⁵, 1×10⁴, 1×10³, and1×10² KG-1a cells in a population of MBA-1 cells (1×10⁶) per tube intriplicates (Table 1). In this example Pd will reflect the number ofKG-1a cells and the presence of surface marker CD34 will specify thecell type, while MBA-1 cells are marked by Rh. Immunolabeling wascarried out by adding anti-CD34 antibodies to tubes containingdecreasing amounts of live KG-1a cells and incubating for 45 minutes onice. Negative control tubes were reacted with the IgG1 isotype controlimmunoglobulins. After three low speed centrifugation washes withPBS/10% FBS all samples were incubated with biotinylated anti-mouseantibodies. This was followed by several washes, and Eu-labeledstreptavidin (DELFIA, Perkin Elmer) was added to all samples as the laststep in the immunolabeling procedure. Un-reacted Eu-strepavidin waseliminated by three final washes. Cell pellets were then dissolvedovernight in 75 ul of concentrated HCl. Prior to ICP-MS analysis, anequal volume of 1 ppb iridium standard (Ir) was added to each sample.Three independent experiments were performed. ICP-MS acquisition of Eu,Rh and Pd signals are presented in FIG. 1A and FIG. 1B. In this example,the detection limit for an abundant biomarker (CD34) is ˜1000 cells inthe presence of 1×10⁶ unrelated cells. Therefore, passive metalaccumulation by cells can be used for normalization of a biomarkerconcentration in solution and particle elemental analysis.

TABLE 1 Samples containing mixed populations of cells cultured in mediasupplemented with palladium (Pd) or rhodium (Rh). Sample KG1-a (Pd )MBA-1 (Rh) #1, 1o 1e6 0 #2, 2o 5e5 5e5 #3, 3o 1e5 1e6 #4, 4o 1e4 1e6 #5,5o 1e3 1e6 #7, 7o 0 1e6

Although antibodies were used in the above example, it is to beunderstood that any affinity product can be used in place of theantibody as is known to those skilled in the art. Further it is possibleto analyze more than one analyte/antigen by using more than one antibodyor affinity product.

Although the analyte/antigen was found on the surface of the cell in theabove example, it is to be understood that analytes/antigens can also befound within the cell and methods for the analysis of these internalanalytes/antigens are included in this invention.

Experiment 2

Yet another embodiment can be demonstrated by the example ofrhodium-containing (Rh(III)) metallointercalator binding to cells.Binding of an intercalator to DNA is governed by the law of mass action.Assuming that a mammalian cell in G1 phase has 3.3×10⁹ DNA base pairs ofwhich only 10-70% are free from protein interaction (nucleosomes) andavailable for binding with the intercalator (one molecule per two basepairs at saturation), the number of molecules of dye needed isapproximately 1-10×10⁸. Thus for [Rh(phi)₂bpy]Cl₃, with 671 MW, 1 ml of200 uM solution should be 100-fold excess of intercalator over DNAbinding sites for 1×10⁶ cells. The binding of various amounts of theRh(III) intercalator with cultured cells was assessed. MBA-1 cells werefixed in 3.7% formaldehyde/PBS for 15 minutes, followed bypermeabilization with 0.3% TRITON™ X-100/PBS; washed once with PBS andincubated for 45 minutes with 1 ml of increasing concentrations of[Rh(phi)₂bpy]Cl₃ from 2.5 nanoM to 250 microM. Each sample contained1×10⁶ cells, run in triplicates. Following washing, the cell pelletswere treated with concentrated HCl, mixed with an equal volume of 1 ppbIr internal standard, and analyzed in solution by ICP-MS. FIG. 2 shows alinear normalized signal response. Therefore, it is observed that theintercalator can bind to DNA in a very broad range of concentrationswithout reaching a saturation limit.

Reactivity of the Rh(III) metallointercalator with fixed andfixed/permeabilized versus live cells was also examined. Equal amountsof MBA-1 cells (1×10⁶) were either fixed/permeabilized as above, fixedin 3.7% formaldehyde alone or left untreated (live cells). After twowashes in PBS the cells were incubated with 200 uM [Rh(phi)₂bpy]Cl₃ for30 minutes; washed three more times and dissolved in HCl. FIG. 3 shows arepresentative graph of two experiments. As evident from FIG. 3, in livecells the Rh(III) complex is membrane impermeable, while fixation andfixation/permeabilization permit DNA and metal complex intercalation tosimilar levels. Therefore, it is observed that non-specific binding ofthe intercalator to the cell surface is very limited; the complex is notinternalized by live cells and the concentration of the Rh(III) complexcorresponds to DNA content. Exact ratio of the intercalator to DNAcontent was investigated in following experiments.

Experiment 3

Yet another embodiment can be demonstrated by example of DNA labelingwith Rh(III) metallointercalator and antigen CD33 surface labeling usingsolution and particle elemental analysis as shown in FIG. 4 and FIG. 9.

MBA-1 cells were fixed/permeabilized with 3.7% formaldehyde and 0.3%TRITON™ X-100 and labeled with Rh(III) intercalator (250 microM).Triplicate samples with 3×c10⁴, 3×10⁵, and 3×10⁶ cells per tube wereprepared. Cells were then incubated with the primary anti-CD33 antibody,followed by secondary Eu-labeled anti-mouse antibody. Cells were washedin 0.65 um centrifugal spin filters (DURAPORE™). After the final wash,filter units were placed over new eppendorf tubes and 80 microL ofconcentrated HCl were added. Overnight digestion of cellular materialwas followed by centrifugation and the filtrate was collected, combinedwith equal volume of Ir internal standard and subjected to ICP-MS. FIG.4 shows simultaneous identification of Rh (representing cell numbers)and Eu (representing cell surface antigen CD33) by solution elementalanalysis.

Experiment 4

In another embodiment, DNA bound to Rh(III) intercalator and surfaceantigen expression were detected in single cells by particle elementalanalysis with ICP-MS operated in transient mode (for flow chart see FIG.9). For these experiments, KG-1a cells were treated with the Rh(III)intercalator as described above. The CD34 surface antigen was detectedvia primary anti-CD34 antibody and secondary gold (Au) labeledanti-mouse antibody. A cell suspension of 1×10⁵ cells/ml was prepared in50 mM ammonium bicarbonate buffer, pH7.4. The suspension was aspiratedinto the ion source of Elan6100 ICP-MS at a rate of 40 microliters perminute, resulting in an average rate of 67 cells per second. Two sets ofexperiments were conducted: one with the mass analyzer set to m/z=103for Rh⁺ detection, another—with m/z=197 for Au⁺ detection. The detectorwas operated in the transient mode, e.g. the ion signals were detectedfrom an output of the analog stage amplifier by storage oscilloscope (HP54610B, Hewlett Packard), and the recorded signals were transferred to acomputer for processing. FIG. 5 shows the examples of data collected forKG-1a cells, with analyzer set to m/z=103 (FIG. 5A) and to m/z=197 (FIG.5B). In FIG. 5A, signals of Rh+ recorded for 16 Rh-positive Au-positivecells are compared to the averaged (n=4) signal for control Rh-negativeAu-positive cells. Two kinds of transients were observed: morefrequently appearing relatively short higher amplitude (70-110microsecond full width at a half maximum) and less frequent long (>150microsecond) with some fine structure and lower amplitude. The totalnumber of ions recalculated from the areas of the peaks (afteraccounting for detector and amplifier gains) is similar between shortand long transients, averaging at 500+−180 Rh⁺ ions per transient. Thenumber of ions in the transient should scale with the number ofintracellular Rh(III) labels, thus providing an evaluation of thecellular DNA and cell size. Experiments (not shown) with larger K562cells (ca. 25 micrometer size compared to 10 micrometer for KG-1a) haveshown that the transients contained on average 5600 Rh⁺ ions. Detectionof Au+ label from Rh-positive Au-positive KG-1a cells is shown in FIG.5B. The signals are compared to the background of Au⁺ signal measuredafter the sample introduction system was rinsed and washed-out for 20minutes with 2% HCl (no Au⁺ could be detected for the controlRh-positive Au-negative cells introduced before). The transient areasaverage at 72+−17 Au+ ions per peak.

Experiment 5

Yet another embodiment can be demonstrated by example of the DNA contentin different cell cycle phases determined with metallointercalator (seeflow chart of FIG. 8). Cells were synchronized in G1/S and G2/M phasesof the cell cycle using a DNA synthesis inhibitor, thymidine, followedby a mitotic inhibitor, nocodazole. The DNA content of the G0/G1 phaseis centered at 2n. Treatment with double thymidine results in aG1/S-phase arrested cell population with approximately 3n DNA content.Nocodazole addition following a single thymidine treatment arrests cellsin the G2/M stage with 4n DNA content. K562 cells were treated with 2 mMthymidine added to culture medium for 17 hours. The cells were thenwashed and incubated in fresh media for 9 hours. At the end of this restperiod the culture was split into two flasks—one received the seconddose of 2 mM thymidine (double thymidine block) to arrest cells at G1/S;the other—0.2 ug/ml nocodazole (single thymidine-nocodazole block) toarrest cells at the M phase. The inhibitors were left in culture mediafor 17 hours. Finally, cells were collected, washed by centrifugationand prepared for DNA staining with propidium iodide (PI) or[Rh(phi)₂bpy]Cl₃. Cells for PI staining were fixed in 75% ethanol at−20° C. for several hours and incubated with 10 microg/ml PI in 1.1%sodium citrate buffer with 1 mg/ml RNase according to standard protocol.Flow cytometry was done on FACSCalibur™ (BD Biosciences) and the resultsconfirmed that after the double thymidine block the majority of cellswere in G1/S, and after the M block practically all cells were in Mphase. For Rh(III) complex staining the cells were fixed in 3.7%formaldehyde, washed and incubated in 100 uM [Rh(phi)₂bpy]Cl₃ solution.After several washes the cells were digested in concentrated HCl andanalyzed by ICP-MS. FIG. 6 illustrates content of DNA (normalizedresponse) depending on the cell cycle phase: mitotic (M), synthetic(G1/S) or normal growth (UN). Moreover, the binding of intercalatingmetal compound was influenced by 10 U/ml Proteinase K (P) or 1 mg/mlRNAse A (R) digestion of stained cells. As evident from the graph,unsynchronized cells have considerably less DNA than mitotic cells,which have approximately two-fold higher amounts of nucleic acid thanG1/S phase cells. Treatment of stained cells with enzymes shifts thedetected Rh values due to enhanced binding of metallointercalator tonaked DNA. As seen in FIG. 6, the amount of Rh is higher in enzymetreated samples R and P for cells in the G1/S phase and unsynchronizedcultures (UN) compared to nontreated control cultures (non). This may bethe result of protease degradation of DNA binding proteins and hencemore nucleic acid being exposed to binding with the metallointercalatorfor P samples. In the case of RNAse A treatment—degradation of small RNAmolecules bound to DNA such as siRNA that may block interaction ofintercalator and DNA may lead to higher detected Rh values. On the otherhand, during mitosis, when DNA is tightly packed into nucleosomes withinthe mitotic chromosomes enzymes fail to uncover “naked DNA” and thisresults in lower Rh signals.

Although ICP-MS was used in the above examples, it is to be understoodthat other forms of elemental analysis are encompassed in theapplicant's teachings.

Kits.

Also provided are kits comprising the components for the analysis byelemental mass spectrometry for the methods described above.

For example, a kit is provided for the method of example 1 for thedetection and measurement of elements, for example rare earth ortransition elements in a sample, where the measured elements originatedfrom an element passively accumulated by cells. The kit can comprise asoluble compound of the element for addition to a cell growth media. Thekit may further comprise instructions for i) spiking a cell growth mediawith the soluble compound of an element, ii) separating bound materialfrom unbound material, and iii) detecting and measuring the passivelyaccumulated element by elemental analysis.

A kit is also provided for the detection and measurement of an element,for example a rare earth or transition element, in a sample, where themeasured elements originated from an element tag on an analyte specificaffinity product that binds with at least one of intra- and/orextra-cellular analyte or analyte complex as described in example 1. Thekit can comprise an element tag comprising the element for directlytagging an analyte specific antibody. The kit can further compriseinstructions for (i) directly tagging an analyte specific affinityproduct; (ii) combining the tagged analyte specific affinity productwith at least one type of an analyte and analyte complex underconditions in which the tagged analyte specific affinity product bindswith at least one of intra- or/and extra-cellular analyte and analytecomplex, (iii) separating bound material from unbound material, and iv)detecting and measuring the element tag by elemental analysis.

Also provided is a kit comprising the combination of the components ofthe kits described above for the passive accumulation of elements by acell and for an affinity product labeled with an element, wherein theaffinity product is specific to an analyte in a cell or on the cellsurface. The kit may further comprise an analyte specific affinityproduct, wherein the analyte specific affinity product is directlytagged with an element tag.

Also provided is a kit for the detection and measurement of an element,for example a rare earth or transition element in a sample, where themeasured elements originated from a metal containing DNA intercalatorfor the method described in example 2. The kit can comprise a metalcontaining intercalator, and optionally instructions for i) conditionsappropriate to incorporate the element containing intercalator into theDNA within the cells of the sample, ii) separating bound material fromunbound material, and iii) detecting and measuring the elementcontaining intercalator by elemental analysis. Also provided is acombination kit comprising these components and the components for thekit for the method of detecting an analyte in or on a cell using aelement labeled affinity product. Further, the kit can comprise ananalyte specific affinity product, wherein the analyte specific affinityproduct is directly tagged with an element tag.

For any of the kits described above, the element can be measured usingan ICP-mass spectrometer. The element can be an isotope or ion. Theelement can be selected from a group consisting of the transitionselements, noble metals, lanthanides, rare earth elements, gold, silver,platinum, rhodium, iridium and palladium. The affinity product can beselected from the group consisting of Fab′, aptamer, antigen, hormone,growth factor, receptor, protein, SH2 peptides and nucleic acid. Theelement can include more than one atom of an isotope. The element caninclude a different number of atoms of each isotope.

Any of the kits can further comprise standards, a dilution buffer, anelution buffer, a wash buffer, and/or an assay buffer. Further, the kitscan comprise two or more elemental tags for simultaneous determinationof two or more analytes. The analyte can be a cytokine. A skilledworker, having read the specification, would understand how thesecomponents work in the methods of the present invention.

The kits described above can further comprise instructions for elementalmass analysis by mass spectrometry.

While the applicant's teachings are described in conjunction withvarious embodiments, it is not intended that the applicant's teachingsbe limited to such embodiments. On the contrary, the applicant'steachings encompass various alternatives, modifications, andequivalents, as will be appreciated by those of skill in the art.

All references cited in the disclosure are herein incorporated byreference.

REFERENCE LIST

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1. A method to determine DNA content and antigen content per cell in asample of cells, comprising: (a) fixing and permeabilizing of the cells;(b) incubating the sample of cells with an intercalator tagged with afirst element under conditions appropriate to incorporate theintercalator into the DNA within the cells of the sample; (c) removingunbound intercalator from the cells; (d) incubating antigen specificaffinity products labeled with a second element tag with the cells; (e)removing unbound affinity products from the cells; (f) simultaneouslymeasuring the intercalator tagged with a first element and the affinityproduct labeled with the second element tag by elemental analysis usingan Inductively Coupled Plasma Mass Spectrometer; and (g) calculating theDNA content and antigen content per cell.
 2. The method of claim 1 wherein (d) the antigen specific affinity product is an antibody complexcomprising a secondary antibody labeled with an element tag that bindsto a primary antibody, and wherein the primary antibody is specific tothe antigen.
 3. The method of claim 1 wherein (d) the cells areincubated with at least two classes of antibodies, each class ofantibodies specific to different classes of antigens and each class ofantibody is labeled with unique element tag.
 4. The method of claim 1further comprising a step after step (d) wherein the cells are dissolvedto generate a relatively homogeneous sample before determining theamount of metal by performing solution elemental analysis.
 5. The methodof claim 4 wherein the cells are dissolved in an acid solution.